|Year : 2012 | Volume
| Issue : 1 | Page : 29-33
Insignificant anti-acne activity of Azadirachta indica leaves and bark
Pratibha Nand1, Sushma Drabu1, Rajinder K Gupta2
1 Maharaja Surajmal Institute of Pharmacy, Janakpuri, India
2 University School of Biotechnology, Guru Gobind Singh Indraprastha University, New Delhi, India
|Date of Web Publication||11-Aug-2012|
Rajinder K Gupta
University School of Biotechnology, G.G.S. Indraprastha University, Sector 16C, Dwarka, New Delhi
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background and Aim: Azadirachta indica has a long history of medicinal usage in skin ailments due to its antibacterial and anti-inflammatory properties. The present study was undertaken to explore the potential of the leaves and bark of Azadirachta indica in the treatment of acne. Materials and Methods: Monographic analysis of the plant was carried out, followed by the phytochemical screening of the sequential extracts of leaves and bark of Azadirachta indica. The results indicated presence of alkaloids, flavonoids, saponins, terpenes, and tannins. Gas chromatography-mass spectrometry (GC/MS) analysis of the test extracts indicated the presence of many phytoconstituents in dichloromethane and methanolic extracts namely stigmasterol, nimbiol, sugiol, 4-cymene, α-terpinene terpinen-4-ol, and vitamin E. The test extracts were evaluated against acne causing bacteria, namely Staphylococcus aureus (MTCC 96), Staphylococcus epidermidis (MTCC 2639), and Propionibacterium acnes (MTCC *1951), for their in vitro antimicrobial activity, using agar disc diffusion method. Results: Dichloromethane and methanolic extracts of Azadirachta indica showed very little activity against S. aureus and S. epidermidis, but did not show any antimicrobial activity against P. acnes. Conclusion: Despite possessing antibacterial properties, Azadirachta indica showed insignificant effect against acne causing bacteria.
Keywords: Antibacterial activity, Azadirachta indica, GC/MS analysis, monographic analysis, phytochemical screening
|How to cite this article:|
Nand P, Drabu S, Gupta RK. Insignificant anti-acne activity of Azadirachta indica leaves and bark. J Pharm Negative Results 2012;3:29-33
|How to cite this URL:|
Nand P, Drabu S, Gupta RK. Insignificant anti-acne activity of Azadirachta indica leaves and bark. J Pharm Negative Results [serial online] 2012 [cited 2017 Dec 18];3:29-33. Available from: http://www.pnrjournal.com/text.asp?2012/3/1/29/99650
| Introduction|| |
Plants have been used for thousands of years in the treatment of skin ailments, as they have great therapeutic potential as antimicrobial agents. Despite the availability of a number of antibiotic and antibacterial agents, the resistant strains of acne necessitate the search for a new anti-acne agent of plant origin. It is also believed that plant-based products are generally safe, have less side effects, and are available at an affordable price. As the literature shows lack of research concerning the utilization of plant extracts in the treatment of acne, an attempt has been made to evaluate the leaves and bark of Azadirachta indica, which possess antibacterial and anti-inflammatory activity.  This plant belongs to the family Meliaceae and grows widely in India, Pakistan, and other tropical and subtropical regions of the world. A. indica A. Juss (Indian neem), and M. azadirachta (Persian lilac) are two closely related species of Meliaceae.  Neem is an evergreen tree with fairly glabrous leaves. Every part of the tree has some therapeutic value and has been used since antiquity as a household remedy against various ailments. , The thickness of the bark varies according to age and the portion of the tree from where it has been taken. Its external surface is rough and fissured in texture with a rusty-gray colour, while the laminated inner surface is yellowish and foliaceous with longitudinal furrows. The leaves are lanceolated, acute, serrated, 7-8.5 cm long and 1.0-1.7 cm wide with slightly yellowish-green colour and possess a characteristic odour along with a bitter taste.  The bark and leaf extracts have been used as folk medicine to control diseases such as leprosy, blood morbidity, itching, skin ulcer, burning sensation, and respiratory disorders. This extract is also fungistatic, antibacterial, and a general health promoter.  It has been used in the treatment of rheumatism, chronic syphilitic sores, and ulcers.  Chemical investigation, mentioned in the literature, highlighted the presence of more than 135 compounds in the different parts of the plant.  These compounds have been classified into two categories :- isoprenoids and nonisoprenoids. The isoprenoids include diterpenoids and triterpenoids containing limonoids, azadirone, nimbin, nimbolide, salanin, and azadirachtin. Nimbin and nimbiol are the bitter principle constituents of this plant. The nonisoprenoids include proteins (amino acids) and carbohydrates (polysaccharides), sulphur compounds, polyphenolics such as flavonoids and their glycosides, dihydrochalcone, coumarin and tannins, and aliphatic compounds.  Nimbolide has shown antibacterial activity against S. aureus and S. coagulase.  Noticeable improvement was observed when a paste of crushed neem leaves, which is a popular home remedy, was applied on the part of face affected by acne.  The bark provided an antibacterial effect due to the presence of margolone, margolonone, isomargolonone, and so on. Condensed tannins in the bark contain gallic acid, (+) gallocatechin, (-) epicatechin, (+) catechin, and epigallocatechin, of which gallic acid , (-) epicatechin, and catechin are primarily responsible for inhibiting the generation of chemiluminescence by the activated human polymorphonuclear neutrophil (PMN),  indicating that these compounds inhibit oxidative burst of PMN during inflammation. Literature has also revealed the presence of polyphenolic compounds in the bark, which have a powerful antioxidant potential to neutralize the damaging effects of free radicals, and these compounds provide anti-inflammatory activity. The methanolic extract of the leaves provided antipyretic and anti-inflammatory effects in male rabbits.  Based on the beneficial effects of Azadirachta indica, the present study is undertaken to ascertain the antimicrobial potential of neem in the treatment of acne.
| Materials and Methods|| |
Collection and identification of plant material
The leaves and bark of Azadirachta indica were collected from the medicinal gardens in Delhi, authenticated by the National Institute of Science Communication and Information Resources (NISCAIR), Pusa Campus, New Delhi with voucher specimen (NISCAIR/RHM/consult/2008-09/978/09), preserved within the department for future reference.
Monographic analysis of herbs
Individual herbs were evaluated according to their standard specifications mentioned in the Ayurvedic Pharmacopoeia of India. Tests were carried out to check the presence of foreign organic matter, loss on drying, total ash, acid-insoluble ash, alcohol-soluble extractive and water-insoluble extractive values.Preparation of extracts
Shade-dried leaves and bark of the plant (200 g) were pulverized separately and subjected to sequential solvent extraction using the continuous hot extraction (soxhlet) method. The extraction was carried out with different solvents present in their increasing order of polarity, such as petroleum ether (PE), dichloromethane (DCM), and methanol (ME). These extracts were abbreviated as: AILPE, AILDCM, AILME and AIBPE, AIBDCM, and AIBME respectively. Each time, the marc was dried in air at room temperature and later used for extraction with other solvents. All the extracts were evaporated using a rotary evaporator and the percentage yield was recorded. Dried extracts were stored at 4°C in airtight containers for further studies.
Concentrated extracts of Azadirachta indica leaves and bark were subjected to qualitative analysis for screening of alkaloids, cardiac glycosides, saponins, tannins, anthraquinones, terpenes, and flavonoids, by using the methods described by Harborne. 
Gas chromatography-mass spectrometry analysis
Three sequential extracts were analyzed for volatile components with an Agilent 6890N gas chromatograph connected to a 5975B mass-selective detector. The chromatographic separation was done on a capillary column of fused silica HP-5 ms (0.25 mm × 30 m × 0.25 μm). 1 μl of each extract was injected in the split mode (1 : 50), with the injector temperature at 280 0 C. The oven temperature was programmed, starting from 70 °C (1min) at 25 °C/min to 150 °C (0 min) at 3 °C/ min to 200 °C (1 min) at 8°C/min, and finally reaching a temperature of 280 °C (3 min). Helium was used as the carrier gas. The detection was performed in the EI mode with ionization energy of 70 eV, source at 230 °C, and quadruple at 150 °C. The relative percentage amounts of the separated compounds were automatically calculated from the peak areas of the total ion chromatogram. The identity of the components was confirmed by comparing their retention time and spectral data with the corresponding data from the NIST'05 library. 
Antibacterial screening microorganism and media
Aerobic bacteria: Staphylococcus aureus (MTCC 96), Staphylococcus epidermidis (MTCC 2639), and anaerobic bacteria: Propionibacterium acnes (MTCC *1951) were obtained from the Microbial Type Culture Collection Centre, Institute of Microbial Technology, Chandigarh. Fresh cultures of the isolates of aerobic and anaerobic bacteria were suspended in nutrient broth and reinforced clostridium medium, respectively. S. aureus and S. epidermidis cultures were incubated for 24 h at 37 °C and 30 °C, respectively. The P. acnes culture was incubated in an anaerobic chamber at 37°C, consisting of 10% CO 2, 10% H 2, and 80% N 2 , for 48 h.
Evaluation of antibacterial activity
The antibacterial activity of extracts was tested using agar disc diffusion method.  100 μl of fresh culture suspension of test bacteria was evenly spread on nutrient agar and reinforced clostridial agar plates. The concentration of cultures was 5 x 10 5 CFU/ml. For screening, a 6 mm diameter filter paper disc, impregnated with 20 μl of extract solution, equivalent to 0.2 mg of extract was placed on the surface of the inoculated media agar plates. Incubation was done at 37 °C and 30 °C for 24 h and 48 h respectively, depending on the type of bacteria, under optimum conditions. Clear zones of inhibition were measured in mm, including the diameter of the disc. The zone measuring 10 mm or more was considered as an effective extract against test organisms. Clindamycin (10 μg/disc) was used as a positive control and the respective solvents used for extraction, served as the negative control.
All the samples for antimicrobial screening were run in triplicate and the mean values were used for the result analysis. Data was expressed as mean ± SEM. Statistical analysis using one way ANOVA was done with SPSS software version 10.0.1 and P values less than 0.05 were considered significant.
| Results|| |
In the present study, the monographic analysis of A. indica leaves and bark revealed that all the tested parameters were within the pharmacopoeial limits [Table 1]. The percentage yield of test extracts indicated that AIBME has the highest percentage yield (11.87) followed by AILME (7.93). Qualitative phytochemical analysis of crude plant extracts revealed the presence of alkaloids, flavonoids, saponins, terpenes, and tannins in the A. indica leaves as well as bark [Table 2]. The previous findings had reported the beneficial effects of flavonoids in fighting ailments and improving human health.  The composition of all the test extracts, after GC/MS analysis, was recorded [Table 3]. Twenty-six important constituents were identified in six test extracts, out of which AILME indicated the presence of stigmasterol (4.55%), phytol(0.9%), 4-cymene(0.73%), α-terpinene(0.9%), and terpinen-4-ol (2.19%).
Vitamin E was found in AILDCM extract. Furthermore, AIBDCM revealed the presence of many active phytoconstituents like stigmasterol (0.44%) and phenolic components, namely, nimbiol (4.77%) and sugiol (1.48%). It also showed the presence of 4-cymene (1.06%), α-terpinene (2.34%), and terpinen-4-ol (4.06%). The antimicrobial screening was carried out on the sequential extracts, using clindamycin phosphate as a positive control [Table 4]. Among these, the highest zone of inhibition was observed in AIBDCM (8.83 ± 0.15 mm) against S. aureus. AILME was found to be effective against the aerobic test strains with inhibition zones of 8.10 ± 0.12 mm and 8.13 ± 0.12 mm against S. aureus and S. epidermidis, respectively. This revealed that the extracts exhibited little activity against test organisms, in comparison to clindamycin (18 ± 0.11 mm). Moreover, AILPE and AIBPE did not show any kind of activity against the test organisms. Also, none of the extracts were found to be effective against P. acnes.
|Table 4: Mean ± SEM (mm) of zone of growth inhibitions, in mm , obtained by preliminary antimicrobial screening of plants against Staphylococcus aureus (MTCC 96), Staphylococcus epidermidis C 2639) and Propionibacterium acnes using the disc diffusion method|
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| Discussion|| |
Neem is a versatile medicinal plant and is a unique source of various compounds possessing diverse therapeutic benefits. In the present study, the phytochemical and GC/MS analyses have indicated the presence of bioactive compounds. Literature has also revealed that the bark and leaves possess polyphenolic compounds, which are responsible for antibacterial, antioxidant, and anti-inflammatory activities.  Another study indicates that petroleum ether, water, and ethanolic extracts of Azadirachta indica exhibit moderate inhibitory activity against P.acnes. In this study, although the active components documented for anti-acne activity, namely α-terpinene and terpinen-4-ol,  were found in both AILME and AIBDCM, the results demonstrated an insignificant anti-acne activity in AILME and AIBDCM extracts, compared to clindamycin.
| Conclusion|| |
Hence, the present study does not support the usage of this plant for the treatment of acne vulgaris because of its insignificant activity against acne-causing organisms.
| Acknowledgments|| |
We acknowledge the financial support from All India Council for Technical Education 8023/BOR/RID/RPS-27/2008-09 & UGC-SAP-3-23/2011. We also would like to thank Professor Rama Choudhary, HOD, Microbiology, All India Institute of Medical Sciences for providing anaerobic microbial facility to carry out antibacterial activity of plant extracts.
| References|| |
|1.||Das BK, Mukherjee SC, Sahu BB, Murjani G. Neem (Azadirachta indica) extract as an antibacterial agent against fish pathogenic bacteria. Indian J Exp Biol 1999;37:1097-100. |
|2.||Chopra RN, Nayer SL, Chopra IC. Glossary of Indian medicinal plants. New Delhi: Council of Scientific and Industrial Research SIR; 1956. p. 31. |
|3.||Koul O, Isman MB, Ketkar CM. Properties and uses of Neem, Azadirachta indica. Can J Bot 1990;68:1-11. |
|4.||Van der Nat JM, Van der Sluis WG, Hart LA, Van Disk H, de Silva KT, Labadie RP. Activity of guided isolation and identification of Azadirachta indica A.Juss. (Meliaceae) bark extract constituents, which specifically inhibit human polymorh nuclear leucocytes. Planta Med 1991;57:65-8. |
|5.||Ayurvedic Pharmacopoeia of India, Govt. of India, New Delhi 1989. Part 1, Vol. 2. p. 131-5. |
|6.||Subapriya R, Nagini S. Medicinal properties of neem leaves: A review. Curr Med Chem Anticancer Agents 2005;5:149-56. |
|7.||Kirtikar KR, Basu BD. Medicinal Plants .Blatter E, Cains JF, Mhaskar KS, editors. Vivek Vihar ,New Delhi: 1975. p. 536. |
|8.||Kausik B, Chattopadhyay I, Banerjee RK, Bandyopadhyay U. Biological activities and medicinal properties of neem (Azadirachta indica). Curr Sci 2002;82:1336-45. |
|9.||Govindachari TR. Chemical and biological investigations on Azadirachta indica (The neem tree). Curr Sci 1992;63:117-22. |
|10.||Biswas K, Chattopadhyay I, Banerjee RK. Biological activities and medicinal properties of neem (Azadirachta indica). Curr Sci 2002;82:1336-45. |
|11.||Ara I, Siddiqui BS, Faizi S, Siddiqui SJ. Structural novel diterpenoid constituents from stem bark of Azadirachta indica (Meliaceae). J Chem Soc Perkin Trans 1989;1:343-5. |
|12.||Vohra SB, Dandiya PC. Herbal analgesic drugs. Fitoterapia 1992;63:195-207. |
|13.||Harbone JB. Phytochemical methods: A guide to modern techniques of plant analysis. London: Chapman and Hall; 1973. p. 279. |
|14.||Adams RP. Identification of essential oils by gas chromatography / Mass spectrometry. 4 th ed. Carol Stream, IL: Allured Publ. Corp; 2007. p. 302. |
|15.||Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-6. |
|16.||Jouad H, Lacaille Dubois MA, Lyoussi B, Eddouks M. Effects of the flavonoids extracted from Spergularia purpurea Pers on arterial blood pressure and renal function in normal and hypertensive rats. J Ethnopharmacol 2001;76:159-63. |
|17.||Van der Nat JM, van der Sluis WG, de Silva KT, Labadie RP. Ethnopharmacognostical survey of Azadirachta indica A Juss (Meliaceae). J Ethnopharmacol 1991;35:1-24. |
|18.||Balakrishnan KP, Narayanaswamy N, Subba P, Poornima EH. Antibacterial activity of certain medicinal plants against acne-inducing bacteria. Int J Pharm Bio Sci 2011;3:476-81. |
|19.||Carson CF, Riley TV. A review: Antimicrobial activity of the essential oil of Melaleuca alternifolia. Lett Appl Microbiol 1993;16:49-55. |
[Table 1], [Table 2], [Table 3], [Table 4]